Transcriptome Sequencing (RNA-Seq) for Identification of Novel Markers of Disease Outcome and Therapeutic Targets in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) represents a heterogeneous group of malignancies with great variability in clinical course and response to therapy. Currently, cytogenetics is the most important prognostic factor in this disease. In recent years, an increasing list of molecular markers with prognostic significance in AML has been identified; nonetheless, new prognostic markers and therapeutic targets are still needed. RNA splicing is a modification of RNA after transcription, in which introns are removed and exons are joined before it can be used to produce a correct protein through translation. Alternative splicing is a process by which the exons of the RNA are reconnected in multiple ways during RNA splicing. The resulting RNA variants may be translated into different proteins with distinct functions. There are bioinformatics and experimental evidence that cancerous cells express transcript variants that are abnormally spliced, suggesting that RNAs are more frequently alternatively spliced in cancerous tissues than in normal ones. Transcriptome sequencing (RNA-Seq) is a new sequencing-based technology that allows the entire transcriptome (set of all RNA molecules) to be survey in an unbiased and high-throughput way allowing identification of novel translocations and splice variants. The goal of the proposed project is to identify (using RNA-Seq), verify (experimentally) and validate (by performing statistical correlation with clinical outcome) novel transcript variants in pediatric AML patients, and to use this knowledge to improve our understanding of human leukemogenesis, develop new molecular classifiers and identify potential therapeutic targets for patients with this highly resistant disease.

Progress Reports and Updates

Project Update

Fabiana Ostronoff answered questions about her research (September 2014):

What were you initially studying with your grant funded by ALSF?
Acute myeloid leukemia (AML) is a heterogeneous group of malignancies with great variability in clinical course and response to therapy. The disease affects all group ages, ranging from newborns to the elderly. Although there has been significant improvement in AML treatment, survival for adolescents and young adults (AYA) has not improved to the same extent as for children. There are likely many reasons to explain the disparity in outcome between AYA and children with AML. Recent studies showed that there are a greater proportion of AYA patients with poor-risk disease relative to younger patients suggesting that, at least in part, the disparity in outcome may be related to different biology of the disease in this age group. In recent years an increasing list of biomarkers with prognostic significance has been identified in AML; nonetheless new biomarkers and potential therapeutic targets among AYA patients are lacking. In this project, we used transcriptome sequencing (RNA-Sequencing) as an unbiased and high-throughput tool to identify novel biomarkers that may be associated with AML pathogenesis in this age group.

What have you found?
Our studies show the applicability of RNA-Sequencing as a tool to discover novel disease-specific markers and potential therapeutic targets in children, adolescents and young adults (CAYA) with acute myeloid leukemia (AML). In this project we identify several alterations in the Nucleoporin (NUP) family of genes, including fusions transcripts, deletions, insertions and copy number alterations. We demonstrated that alterations in NUPs, such as NUP98/NSD1, have independent prognostic value in CAYA patients with AML. Patients harboring this genetic alteration have a disease highly resistant to conventional chemotherapy and would benefit from alternative therapeutic approaches upfront. Additional data from this project suggest that other NUP alterations are prevalent in AML and are likely associated with disease resistance.

What does this mean for children with cancer and their families?
This project unveiled a potential entire new class of biological markers in AML that are especially prevalent in pediatric and young adults with AML. The characterization of these novel alterations in the Nucleoporin family of genes will likely define a new class of molecular markers in AML, which will improve disease risk stratification and therapy allocation. In addition, unveiling the role of NUP alterations in AML will not only enhance our understanding of the mechanism of leukemia transformation and disease resistance but also open opportunities for more effective targeted therapy for a significant number of patients harboring these genetic alterations.

What are your next steps?
Our future plans include completing the characterization of these novel alterations and correlating the presence of these NUP alterations with clinical outcomes in CAYA patients with AML. Our data suggest that Nucleoporin alterations are associated with disease resistance. Currently, there is no biological marker that is able to predict disease resistance in AML. If these alterations are shown to predict disease resistance, these markers will be incorporated in future Children Oncology Group (COG) trials for therapy. We will investigate the pathways that are deregulated in AML associated with NUP alterations, which will not only improve our understanding of the biology of the disease in this subgroup of patients but also potentially identify targets for more effective therapies. Finally, we have optimized a high-throughput drug screening to investigate over 500-targeted agents for selective activity against cells harboring NUP alterations. Using this platform we will likely identify effective agents for the treatment of patients harboring these alterations.

Has this research been published?
Yes, it was published in Blood. Ostronoff F et al. Co-expression of NUP98/NSD1 and FLT3/ITD is more prevalent in younger AML patients and leads to high-risk of induction failure: a COG and SWOG report [in press].
This work was also presented at the American Society of Hematology Annual Meetings:

Ostronoff F, Othus M, Gerbing R, et al. NUP98/NSD1 translocation further risk-stratifies patients with FLT3/ITD acute myeloid leukemia: a report from Children’s Oncology Group. Blood (ASH Annual Meeting, 2013) [Oral]
Ostronoff F, Alonzo TA, Gerbing R, Loken M, Pardo L et al. Cryptic NUP98/NSD1 Translocations Are Highly Prevalent in FLT3/ITD-Positive Acute Myeloid Leukemia and Lead to High Rate of Induction Failure. Report From Children’s Oncology Group. Blood (ASH Annual Meeting, 2012) [Oral]
Ostronoff F, Fitzgibbon M, McIntosh M, Ries R, Gamis AS et al. RNA-Sequencing Unveils Cryptic Fusions in Patients with Acute Myeloid Leukemia. Blood (ASH Annual Meeting, 2012) [Poster]

What has this grant from ALSF allowed you to do that you wouldn’t have been able to do otherwise?
The Alex’s Lemonade Young Investigator Award allowed me the necessary protected time to execute my experiments and develop my career as an independent investigator.

Why did you choose to work in this field/on this topic?
AML is a genetically heterogeneous and highly resistant hematopoietic malignancy. Despite the growing number of molecular markers that have been identified in AML, there has been very little therapeutic progress in this disease in decades with the majority of patients still dying from their disease. This lack of therapeutic advancement is in part due to the paucity of actionable biological targets in this disease. My goal is to improve our understanding of the biology of this disease and apply our findings to improve outcome of these patients.

How did you benefit from being mentored during this project; what was most helpful?
Dr. Meshinchi is an outstanding mentor. He has great enthusiasm for our research, extensive knowledge and is a recognized leader in the filed. He has shown a strong commitment to my career development and made available all resources for the successful execution of this project. Dr. Meshinchi has provided guidance and support throughout this project, while helping me to transition to become an independent investigator.

From Dr. Ostronoff's mentor, Soheil Meshinchi, MD, PhD, on the benefits of mentoring her:
It has been a great pleasure mentoring Dr. Ostornoff. She is a very talented and dedicated young investigator. During the two-year period of this award, Dr. Ostronoff has generated enormous amount of data and made major contributions to the field of AML. She has characterized a large number of novel alterations in the Nucleoporin family of genes, which are likely to define a new class of molecular markers and identify new therapeutic targets in this disease. The key activities for Dr. Ostronoff’s development as an independent investigator included her bench research work, analysis and interpretation of her data, presenting her work at national meetings such as the American Society of Hematology, writing manuscripts and grant applications. Dr. Ostronoff has been very productive and has done exceedingly well. I am confident she will become a successful independent investigator. It has been a pleasure working with her and being her mentor.

Institutions